The assessment of genetically modified (GM) crops for regulatory approval currently requires a detailed molecular characterization of the DNA sequence and integrity of the transgene locus. In addition, molecular characterization is a critical component of event selection and advancement during product development. Typically, molecular characterization has relied on Southern blot analysis to establish locus and copy number along with targeted sequencing of polymerase chain reaction products spanning any inserted DNA to complete the characterization process. Here we describe the use of next generation (NexGen) sequencing and junction sequence analysis bioinformatics in a new method for achieving full molecular characterization of a GM event without the need for Southern blot analysis. In this study, we examine a typical GM soybean [Glycine max (L.) Merr.] line and demonstrate that this new method provides molecular characterization equivalent to the current Southern blot‐based method. We also examine an event containing in vivo DNA rearrangement of multiple transfer DNA inserts to demonstrate that the new method is effective at identifying complex cases. Next generation sequencing and bioinformatics offers certain advantages over current approaches, most notably the simplicity, efficiency, and consistency of the method, and provides a viable alternative for efficiently and robustly achieving molecular characterization of GM crops.
Small RNAs (∼20 to 24 nucleotides) function as naturally occurring molecules critical in developmental pathways in plants and animals [1], [2]. Here we analyze small RNA populations from mature rice grain and seedlings by pyrosequencing. Using a clustering algorithm to locate regions producing small RNAs, we classified hotspots of small RNA generation within the genome. Hotspots here are defined as 1 kb regions within which small RNAs are significantly overproduced relative to the rest of the genome. Hotspots were identified to facilitate characterization of different categories of small RNA regulatory elements. Included in the hotspots, we found known members of 23 miRNA families representing 92 genes, one trans acting siRNA (ta-siRNA) gene, novel siRNA-generating coding genes and phased siRNA generating genes. Interestingly, over 20% of the small RNA population in grain came from a single foldback structure, which generated eight phased 21-nt siRNAs. This is reminiscent of a newly arising miRNA derived from duplication of progenitor genes [3], [4]. Our results provide data identifying distinct populations of small RNAs, including phased small RNAs, in mature grain to facilitate characterization of small regulatory RNA expression in monocot species.
The DNA sequence that encodes 23S rRNA domain V of Bacilus subtilis, nucleotides 2036 to 2672 (C. J. Green, G. C. Stewart, M. A. Hollis, B. S. Vold, and K. F. Bott, Gene 37:261-266, 1985), was cloned and used as a template from which to transcribe defined domain V RNA in vitro. The RNA transcripts served as a substrate in vitro for specific methylation of B. subtilis adenine 2085 (adenine 2058 in Escherichia coli 23S rRNA) by the ErmSF methyltransferase, an enzyme that confers resistance to the macrolide-lincosamidestreptogramin B group of antibiotics on Streptomycesfiradiae NRRL 2702, the host from which it was cloned.Thus, neither RNA sequences belonging to domains other than V nor the association of 23S rRNA with ribosomal proteins is needed for the specific methylation of adenine that confers resistance to the macrolide-lincosamide-streptogramin B group of antibiotics.The ern N-methyltransferases (methylases) constitute a group of resistance factor enzymes that confer resistance to the macrolide, lincosamide, and streptogramin B antibiotics by methylating a specific adenine residue in 23S rRNA within the sequence GAAAG (11)(12)(13)(14). The precise location of the methylated adenine residue in Bacillus stearothermophilus 23S rRNA corresponds to that of adenine 2058 (A2058) in Escherichia coli 23S rRNA, as shown by Skinner et al. (23), and this location, in turn, corresponds to that of A2085 of Bacillus subtilis 23S rRNA, whose sequence was determined by Green et al. (6). Pulse-chase experiments with labeled adenosine (11) reveal that the methylase utilizes nascent 23S rRNA rather than mature 50S subunits as the substrate. Pulse-chase experiments, however, cannot rule out the possibility that the nascent 23S rRNA is utilized by the methylase in vivo in the form of partially assembled subparticles of the 50S subunit instead. Phenol-extracted 23S rRNA from ribosomes sensitive to macrolide, lincosamide, and streptogramin B antibiotics can serve as a substrate for a partially purified methylase preparation (22); however, the RNA thus prepared could contain an additional active structural component, present in the ribosome from which the rRNA substrate was extracted.The simplest model to describe the requirements for methylation of 23S rRNA postulates that rRNA alone can function as a substrate. If this is so, which sequence characteristics of 23S rRNA confer the specificity that enables its recognition by erm methylases? To evaluate the possibilities would require that a truncated rRNA be used as a substrate for in vitro methylation by a purified enn methylase preparation. MATERUILS AND METHODSStrains, plasmids, and primers. Bacterial strains and plasmids that were used in this study are listed in Table 1.Oligonucleotide primers that were used are listed in Table 2. ermSF cloning and expression in E. coli. PCR was performed (15), with modifications for the use of Streptomyces fradiae NRRL 2702 chromosomal DNA as the template. The two oligonucleotides 8803, a 45-mer, and 8800, a 24-mer, were used as upstream (sen...
ermSF (synonym tlrA) from Streptomyces fradiae NRRL 2702 confers resistance to the macrolide-lincosamide- streptogramin type B (MLS) superfamily of antibiotics. ErmSF specifically methylates Bacillus subtilis 23S rRNA in vitro at A2085 (B. subtilis coordinate, which is equivalent to the Escherichia coli coordinate A2058). In the present studies, partial B. subtilis 23S rRNA sequences containing portions of the peptidyltransferase circle which include A2085 were constructed in order to identify structural requirements needed for RNA to function as substrate of ErmSF. A model methylase substrate based on the 41-nucleotide construct DK111, ggCCUAUCCGUCGCGGGUUCGCCCGCGACAGGACGGA*AAGA, had methyl-acceptor activity. This sequence contains 23S rRNA stem 73 [Stade, K., et al. (1994) Nucleic Acids Res. 22, 1394-1399] underlined, flanking a tetraloop-like (UUCG), and the impaired sequence AAAGA, at the 3' end containing A2085 (A*). A set of systematic alterations introduced into the sequence suggested that the four unpaired nucleotides in stem 73 are necessary for methyl-acceptor activity, whereas inversion of 11 out 13 paired bases in stem 73 conferred no significant reduction in methyl-acceptor activity.
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